Horizontal fissuring at the osteochondral interface: a novel and unique pathological feature in patients with obesity-related osteoarthritis.

OBJECTIVES: Obesity is a well-recognised risk factor for osteoarthritis (OA). Our aim is to characterise body mass index (BMI)-associated pathological changes in the osteochondral unit and determine if obesity is the major causal antecedent of early joint replacement in patients with OA. METHODS: We analysed the correlation between BMI and the age at which patients undergo total knee replacement (TKR) in 41 023 patients from the Australian Orthopaedic Association National Joint Replacement Registry. We then investigated the effect of BMI on pathological changes of the tibia plateau of knee joint in a representative subset of the registry. RESULTS: 57.58% of patients in Australia who had TKR were obese. Patients with overweight, obese class I & II or obese class III received a TKR 1.89, 4.48 and 8.08 years earlier than patients with normal weight, respectively. Microscopic examination revealed that horizontal fissuring at the osteochondral interface was the major pathological feature of obesity-related OA. The frequency of horizontal fissure was strongly associated with increased BMI in the predominant compartment. An increase in one unit of BMI (1 kg/m2) increased the odds of horizontal fissures by 14.7%. 84.4% of the horizontal fissures were attributable to obesity. Reduced cartilage degradation and alteration of subchondral bone microstructure were also associated with increased BMI. CONCLUSIONS: The key pathological feature in OA patients with obesity is horizontal fissuring at the osteochondral unit interface. Obesity is strongly associated with a younger age of first TKR, which may be a result of horizontal fissures.

Measurement and limitations of optical orbital angular momentum through corrected atmospheric turbulence.

In recent years, there have been a series of proposals to exploit the orbital angular momentum (OAM) of light for astronomical applications. The OAM of light potentially represents a new way in which to probe the universe. The study of this property of light entails the development of new instrumentation and problems which must be addressed. One of the key issues is whether we can overcome the loss of the information carried by OAM due to atmospheric turbulence. We experimentally analyze the effect of atmospheric turbulence on the OAM content of a signal over a range of realistic turbulence strengths typical for astronomical observations. With an adaptive optics system we are able to recover up to 89% power in an initial non-zero OAM mode (ℓ = 1) at low turbulence strengths (0.30" FWHM seeing). However, for poorer seeing conditions (1.1" FWHM seeing), the amount of power recovered is significantly lower (5%), showing that for the terrestrial detection of astronomical OAM, a careful design of the adaptive optics system is needed.

DNA damage drives antigen diversification through mosaic Variant Surface Glycoprotein (VSG) formation in Trypanosoma brucei.

Antigenic variation, using large genomic repertoires of antigen-encoding genes, allows pathogens to evade host antibody. Many pathogens, including the African trypanosome Trypanosoma brucei, extend their antigenic repertoire through genomic diversification. While evidence suggests that T. brucei depends on the generation of new variant surface glycoprotein (VSG) genes to maintain a chronic infection, a lack of experimentally tractable tools for studying this process has obscured its underlying mechanisms. Here, we present a highly sensitive targeted sequencing approach for measuring VSG diversification. Using this method, we demonstrate that a Cas9-induced DNA double-strand break within the VSG coding sequence can induce VSG recombination with patterns identical to those observed during infection. These newly generated VSGs are antigenically distinct from parental clones and thus capable of facilitating immune evasion. Together, these results provide insight into the mechanisms of VSG diversification and an experimental framework for studying the evolution of antigen repertoires in pathogenic microbes.

Pathogenesis and clinical management of obesity-related knee osteoarthritis: Impact of mechanical loading.

Obesity-related osteoarthritis (OA) is a complex, multifactorial condition that can cause significant impact on patients' quality of life. Whilst chronic inflammation, adipocytokines and metabolic factors are considered to be important pathogenic factors in obesity related OA, there has been limited investigation into the biomechanical impact of obesity on OA development. This review aims to demonstrate that mechanical factors are the major pathological cause of obesity-related OA. The effect of obesity on pathological changes to the osteochondral unit and surrounding connective tissues in OA is summarized, as well as the impact of obesity-related excessive and abnormal joint loading, concomitant joint malalignment and muscle weakness. An integrated therapeutic strategy based on this multi-factorial presentation is presented, to assist in the management of obesity related OA. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Despite the high prevalence of obesity-related OA, there is no specific guideline available for obesity-related OA management. In this review, we demonstrated the pathological changes of obesity-related OA and summarized the impact of biomechanical factors by proposing a hypothetical model of obesity-related OA change. Therapeutic strategies based on adjusting abnormal mechanical effects are presented to assist in the management of obesity-related OA.

A critical function for beta-amyloid precursor protein in neuronal migration revealed by in utero RNA interference.

Physiological processing of the beta-amyloid precursor protein (APP) generates amyloid beta-protein, which can assemble into oligomers that mediate synaptic failure in Alzheimer's disease. Two decades of research have led to human trials of compounds that chronically target this processing, and yet the normal function of APP in vivo remains unclear. We used the method of in utero electroporation of shRNA constructs into the developing cortex to acutely knock down APP in rodents. This approach revealed that neuronal precursor cells in embryonic cortex require APP to migrate correctly into the nascent cortical plate. cDNAs encoding human APP or its homologues, amyloid precursor-like protein 1 (APLP1) or APLP2, fully rescued the shRNA-mediated migration defect. Analysis of an array of mutations and deletions in APP revealed that both the extracellular and cytoplasmic domains of APP are required for efficient rescue. Whereas knock-down of APP inhibited cortical plate entry, overexpression of APP caused accelerated migration of cells past the cortical plate boundary, confirming that normal APP levels are required for correct neuronal migration. In addition, we found that Disabled-1 (Dab1), an adaptor protein with a well established role in cortical cell migration, acts downstream of APP for this function in cortical plate entry. We conclude that full-length APP functions as an important factor for proper migration of neuronal precursors into the cortical plate during the development of the mammalian brain.

Physiological regulation of the beta-amyloid precursor protein signaling domain by c-Jun N-terminal kinase JNK3 during neuronal differentiation.

Beta-amyloid precursor protein (APP) is a conserved and ubiquitous transmembrane glycoprotein strongly implicated in the pathogenesis of Alzheimer's disease but whose normal biological function is unknown. Analogy to the Notch protein suggests that APP is a cell-surface receptor that signals via sequential proteolytic cleavages that release its intracellular domain (AICD) to the nucleus. Because these cleavages are major targets for therapeutic inhibition, it is critical to elucidate their physiological function. AICD is stabilized by Fe65, interacts with the transcriptional factor Tip60, and translocates to the nucleus. Here, we show that endogenous AICD in primary neurons is detectable only during a short period of time during differentiation in culture. During this transient rise, a portion of AICD localizes to the nucleus. Subsequently, phosphorylation of the APP cytoplasmic domain at threonine 668 appears to disrupt the stabilizing interaction with Fe65 and thus downregulate AICD-mediated signaling. Furthermore, we find that the neuron-specific c-Jun N-terminal kinase JNK3, but not JNK1 or JNK2, mediates a substantial portion of this phosphorylation. We conclude that endogenous AICD undergoes tight temporal regulation during the differentiation of neurons and is negatively regulated by JNK3 via phosphorylation of APP at Thr668.

In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats.

Organ decellularization is emerging as a promising regenerative medicine approach as it is able to provide an acellular, three-dimensional biological scaffold material that can be seeded with living cells for organ reengineering. However this application is currently limited to donor-derived decellularized organs for reengineering in vitro and no study has been conducted for re-engineering the decellularized organ in vivo. We developed a novel technique of a single liver lobe decellularization in vivo in live animals. Using a surgical method to generate a by-pass circulation through the portal vein and infra-hepatic vena cava with a perfusion chamber system, we decellularized the single liver lobe and recellularized it with allogenic primary hepatocytes. Our results showed that the decellularization process in vivo can preserve the vascular structural network and functional characteristics of the native liver lobe. It allows for efficient recellularization of the decellularized liver lobe matrix with allogenic primary hepatocytes. Upon the re-establishment of blood circulation, the recellularized liver lobe is able to gain the function and the allogenic hepatocytes are able to secret albumin. Our findings provide a proof of principle for the in vivo reengineering of liver.

Mapping polycyclic aromatic hydrocarbon and aromatic amine-induced DNA damage in cancer-related genes at the sequence level.

Genomic injury induced by environmental carcinogens, such as polycyclic aromatic hydrocarbons and aromatic amines, is the initial step that can trigger mutagenesis and carcinogenesis. In addition to the physico-chemical property of DNA damaging agents, several important factors such as primary sequence, chromatin structure, methylation, protein association, and transcriptional activity can affect not only the initial level and distribution of DNA damage but also the efficiency of repair. Therefore, mapping the DNA damage induced by environmental agents in cancer-related genes such as p53 and ras at the sequence level provides essential information for assessing their carcinogenic potential. Recently, using the E. coli nucleotide excision enzyme complex, UvrABC nucleases in combination with ligation-mediated polymerase chain reaction, we developed a method to map DNA damage in the p53 and ras genes. These studies led us to conclude that targeted DNA damage, in combination with growth selection, contributes greatly in shaping the mutation spectrum in these genes in human cancer. Here we present the rationale and details of this approach, typical experimental results and necessary precautions.

Novel PAX6 binding sites in the human genome and the role of repetitive elements in the evolution of gene regulation.

Pax6 is a critical transcription factor in the development of the eye, pancreas, and central nervous system. It is composed of two DNA-binding domains, the paired domain (PD), which has two helix-turn-helix (HTH) motifs, and the homeodomain (HD), made up from another HTH motif. Each HTH motif can bind to DNA separately or in combination with the others. We identified three novel binding sites that are specific for the PD and HD domains of human PAX6 from single-copy human genomic DNA libraries using cyclic amplification of protein binding sequences (CAPBS) and electrophoretic mobility shift assays (EMSAs). One of the binding sites was found within sequences of repetitive Alu elements. However, most of the Alu sequences were unable to bind to PAX6 because of a small number of mismatches (mostly in CpG dinucleotide hot spots) in the consensus Alu sequences. PAX6 binding Alu elements are found primarily in old and intermediate-aged Alu subfamilies. These data along with our previously identified B1-type Pax6 binding site showed that evolutionarily conserved Pax6 has target sites that are disparate in primates and rodents. This difference indicates that human and mouse Pax6-regulated gene networks may have evolved through these lineage-specific repeat elements.

Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet.

Amyloid beta (Abeta) protein immunotherapy lowers cerebral Abeta and improves cognition in mouse models of Alzheimer's disease (AD). Here we show that Caribbean vervet monkeys (Chlorocebus aethiops, SK) develop cerebral Abeta plaques with aging and that these deposits are associated with gliosis and neuritic dystrophy. Five aged vervets were immunized with Abeta peptide over 10 months. Plasma and cerebral spinal fluid (CSF) samples were collected periodically from the immunized vervets and five aged controls; one monkey per group expired during the study. By Day 42, immunized animals generated plasma Abeta antibodies that labeled Abeta plaques in human, AD transgenic mouse and vervet brains; bound Abeta1-7; and recognized monomeric and oligomeric Abeta but not full-length amyloid precursor protein nor its C-terminal fragments. Low anti-Abeta titers were detected in CSF. Abetax-40 levels were elevated approximately 2- to 5-fold in plasma and decreased up to 64% in CSF in immunized vervets. Insoluble Abetax-42 was decreased by 66% in brain homogenates of the four immunized animals compared to archival tissues from 13 age-matched control vervets. Abeta42-immunoreactive plaques were detected in frontal cortex in 11 of the 13 control animals, but not in six brain regions examined in each of the four immunized vervets. No T cell response or inflammation was observed. Our study is the first to demonstrate age-related Abeta deposition in the vervet monkey as well as the lowering of cerebral Abeta by Abeta vaccination in a non-human primate. The findings further support Abeta immunotherapy as a potential prevention and treatment of AD.